Insulin-like growth factors (IGFs) mediate differentiation decisions during embryonic development, cell growth signaling and responses to metabolic stress. We found that the production of these important growth factors is dependent on the activity of the endoplasmic reticulum chaperone glucose regulate protein 94 (GRP94), which is essential for their biosynthesis. GRP94 is different from other molecular chaperones in its selectivity towards only a small number of client proteins, preference for late folding intermediates and the absence of any known co-factors. All these aspects suggest a unique mode of action for GRP94. This project will elucidate the enigmatic action cycle of GRP94 by taking advantage of cells whose growth depends on the GRP94-IGF axis. In our first aim we will use a novel cell-based assay to screen for mutants of GRP94 that do not support IGF-II production and thus define amino acids that are essential for this chaperone function. In Aim 2, we will determine whether these mutants are defective in general mechanistic aspects, such as ATP binding and hydrolysis or calcium binding, or whether the mutations define a client binding domain. We will also test the ability of GRP94 mutants to support clients other than IGF, to discover whether there are client-specific mutants. Our third aim is to understand how GRP94 recognizes IGF, by exploiting the structural similarities and differences among a set of insulin-like proteins. These studies will inform on the client selectivity of GRP94, which is currently not understood. Finally, we will take advantage of the clinical consequences of IGF deficiency to search for functionally important human GRP94 variants. Following on our finding that at least one such variant affects IGF production, we will genotype and sequence IGF-deficient patient populations to seek allelic GRP94 variants that may explain low IGF production. Together, these results will lead to a new understanding of how GRP94 chaperones client proteins.